Biological Characteristics And Transcriptome Analysis Of Haemophilus Parasuis Biofilm

Haemophilus parasuis(Hps)is a conditional pathogen that colonizes the upper respiratory tract of pigs,belongs to the buzz deco Haemophilus,is a kind of no motility,NAD dependency of gram-negative bacteria.Gl?sser's disease remains a significant economic burden for the pig industry,with major impact in the nursery and early fattening stages.Like Pseudomonas aeruginosa,Haemophilus parasuis can form a biofilm in vitro,and the amount of biofilm produced by different H.parasuis strains varies.Studies have shown that the formation of H.parasuis biofilms is closely related to the colonization of non-virulent strains,and also contributes to the development of resistance to H.parasuis,which poses a huge challenge to the treatment of Glaser disease.At present,researches on H.parasuis biofilms at home and abroad have mainly focused on the formation mechanism of biofilms and enhanced resistance and tolerance to the host.However,considering only individual proteins and genes,it is not enough to consider H.parasuis biofilms.The in-depth study provides sufficient reference.In order to find H.parasuis biofilm formation related genes,the gene expression regulation mechanism of biofilm formation was studied.This study improved the in vitro culture biofilm model,selected multiple strains of Haemophilus parasuis with strong biofilm formation ability,determined the biofilm formation cycle,measured the enzyme digestion of the biofilm,observed the three-dimensional structure of the biofilm,and used RNA-seq sequencing The technology was used to compare the transcriptome of Haemophilus parasuis strain HPS-W and the biofilm-forming bacteria and planktonic bacteria.The enriched functional entries,biochemical metabolism and signal transduction pathways of differential genes were analyzed by GO and KEGG enrichment.1.Determination and Biological Characteristics of Haemophilus parasuis BiofilmThis study improved the method of culturing biofilms in vitro through cell culture plates.From 13 strains of Haemophilus parasuis,5 strains with strong biofilm formation ability and 4 strains with weak biofilm formation capacity were selected.The amount of biofilm formation increased rapidly in the first 24 hours and stabilized at 72 hours.The biofilms of H.parasuis were most sensitive to Proteinase K treatment,followed by DNAse I treatment,and resistant to Disperin B treatment.The results show that Haemophilus parasuis is mostly short rod-shaped,the bacteria adhere to the surface of the medium,multiple bacteria are bonded and stacked together,and the extracellular matrix surrounds the stacked bacteria to form a membrane-like bacteria polymer.2.Analysis of the role of genes related to biofilm formation of Haemophilus parasuis based on RNA-seq technologyIn the above paper,the Hps-w strain with the strongest biofilm formation ability was selected as the research object,the biofilm and plankton-state bacteria were cultured,and the m RNA transcription of the two samples was detected by rna-seq technology.After 6samples were sequenced,the low-quality reads with adapters were filtered out to obtain valid data.The clean bases totaled 8.31 G.The sequencing error rates did not exceed 0.3%,and the Q30 content was greater than 90%.The GC content was more consistent.The average ratio of valid data reads to the reference genome and reference gene sequence was94.93% and 86.18%,respectively.The gene expression abundance is high,and the number of genes with the highest expression level at log10(FPKM + 1)= 2.Based on readcount data obtained from gene expression level analysis,a total of 128 differentially expressed genes of membrane-state bacteria were screened according to the criteria of qvalue?0.05,|log2Fold Change|?1,and FPKM?1.There are 72 genes expression significantly raised,56 gene expression significantly lowered.GO enrichment analysis showed that the differential genes were mainly involved in biological processes such as protein folding,carbohydrate transport,amino acid metabolism in the glutamine family,and molecular functions such as adenylate exchange factor activity,protein binding,and structure-specific DNA binding.KEGG pathway enrichment analysis showed that the pathways enriched by differential genes mainly involved starch and sucrose metabolism,metabolic pathways,folic acid biosynthesis,ABC transporter and other pathways.In order to verify the reliability of the RNA-seq data,this study used q RT-PCR technology and 16 Sr RNA gene as an internal reference to verify 14 randomly selected differentially expressed genes.The comparison shows that the results of q RT-PCR verification of differentially expressed genes are basically the same as the results of differentially expressed genes in RNA-seq data,indicating the reliability of RNA-seq data.